Epithelial-mesenchymal Transition Of Colorectal Cancer Cells Research Articles

S100A6 mediated epithelial-mesenchymal transition affects chemosensitivity of colorectal cancer to oxaliplatin

PurposeTo investigate the mechanism by which S100 calcium-binding protein A6 (S100A6) affects colorectal cancer (CRC) cells to oxaliplatin (L-OHP) chemotherapy, and to explore new strategies for CRC treatment. MethodsS100A6 expression was assessed in both parental and L-OHP-resistant CRC cells using western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assays (ELISA). Lentiviral vectors were utilized to induce the knockdown of S100A6 expression, followed by comprehensive evaluations of cell proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). Additionally, RNA-seq analysis was conducted to identify genes associated with the knockdown of S100A6. ResultsElevated S100A6 expression in CRC tissues correlated with an adverse prognosis in patients with CRC. Higher expression of S100A6 was also observed in L-OHP-resistant CRC cells, which showed enhanced proliferation, migration, invasion, and antiapoptotic capabilities. Notably, the knockdown of S100A6 expression resulted in decreased proliferation, increased apoptosis, and suppression of EMT and tumorigenicity in L-OHP-resistant CRC cells. Transcriptome sequencing reveals a noteworthy association between S100A6 and vimentin expression. Application of the EMT agonist, transforming growth factor β (TGF-β), induces EMT in CRC cells. S100A6 expression positively correlates with TGF-β expression. TGF-β facilitated the expression of EMT-related molecules and reduced the chemosensitivity of L-OHP in S100A6-knockdown cells. ConclusionIn conclusion, the knockdown of S100A6 may overcome the L-OHP resistance of CRC cells by modulating EMT.

PSAT1 mediated EMT of colorectal cancer cells by regulating Pl3K/AKT signaling pathway.

Background: The metastasis of colorectal cancer (CRC) is one of the significant barriers impeding its treated consequence and bring about high mortality, less surgical resection rate and poor prognosis of CRC patients. PSAT1 is an enzyme involved in serine biosynthesis. The studies showed that PSAT1 plays the part of a crucial character in the regulation of tumor metastasis. And Epithelial-Mesenchymal Transition (EMT) is a process of cell reprogramming in which epithelialcells obtain mesenchymal phenotypes. It is a crucial course in promoting cell metastasis and the progression of malignant tumors. The relationship between PSAT1 and EMT in colorectal cancer, as well as the underlying molecular mechanisms, remains enigmatic and warrants thorough exploration. These findings suggest that PSAT1 may serve as a promising therapeutic target for mitigating colorectal cancer metastasis and holds the potential to emerge as a valuable prognostic biomarker in forthcoming research endeavors. Materials and Methods: Utilizing TCGA dataset in conjunction with clinical CRC specimens, our initial focus was directed towards an in-depth examination of PSAT1 expression within CRC, specifically exploring its potential correlation with the adverse prognostic outcomes experienced by patients. Furthermore, we conducted a comprehensive investigation into the regulatory influence exerted by PSAT1 on CRC through the utilization of siRNA knockdown techniques. In the realm of in vitro experimentation, we meticulously evaluated the impact of PSAT1 on various facets of CRC progression, including cell migration, invasion, proliferation, and colony formation. In order to elucidate the intricate effects in question, we adopted a multifaceted methodology that encompassed a range of assays and analyses. These included wound healing assays, transwell assays, utilization of the Cell Counting Kit-8 (CCK-8) assay, and colony formation assays. By employing this diverse array of investigative techniques, we were able to achieve a comprehensive comprehension of the multifaceted role that PSAT1 plays in the pathogenesis of colorectal cancer. This multifarious analysis greatly contributed to our in-depth understanding of the complex mechanisms at play in colorectal cancer pathogenesis. Using WB and PCR experiments, we found that PSAT1 has a role in regulating EMT development in CRC.In terms of mechanism, we found that PSAT1 affected EMT by Regulating Pl3K/AKT Signaling Pathway. Results: Our investigation revealed a noteworthy down-regulation of PSAT1 expression in CRC specimens. Importantly, this down-regulation exhibited a significant positive correlation with the unfavorable prognosis of patients afflicted with CRC. Functionally, our study showcased that the siRNA-mediated knockdown of PSAT1 markedly enhanced various key aspects of CRC pathogenesis in an in vitro setting. Specifically, this included a substantial promotion of CRC cell migration, invasion, proliferation, and colony formation. Moreover, the silencing of PSAT1 also demonstrated a substantial promotion of the EMT process. Intriguingly, our research unveiled a hitherto unexplored mechanism underlying the regulatory role of PSAT1 in CRC and EMT. We have established, for the first time, that PSAT1 exerts its influence by modulating the activation of the PI3K/AKT Signaling Pathway. This mechanistic insight provides a valuable contribution to the understanding of the molecular underpinnings of CRC progression and EMT induction mediated by PSAT1. Conclusions: In unison, our research findings shed light on the previously uncharted and significant role of the PSAT1/PI3K/AKT axis in the initiation of the EMT process in CRC. Furthermore, our discoveries introduce a novel biomarker with potential implications for the clinical diagnosis and treatment of CRC.

MKRN1 promotes colorectal cancer metastasis by activating the TGF-β signalling pathway through SNIP1 protein degradation

BackgroundThe Makorin ring finger protein 1 (MKRN1) gene, also called RNF61, is located on the long arm of chromosome 7 and is a member of the RING finger protein family. The E3 ubiquitin ligase MKRN1 is closely linked to tumour development, but the exact mechanism needs to be elucidated. In this study, we aimed to investigate the specific mechanism and role of MKRN1 in colorectal cancer (CRC) development.MethodsMKRN1 expression in CRC was analysed using the Cancer Cell Line Encyclopaedia and the Cancer Genome Atlas (TCGA) databases. Rectal tumour tissues were frozen to explore the MKRN1 expression in CRC and its clinical significance. The impact of MKRN1 on CRC cell proliferation and migration was observed using CCK8, colony formation, wound healing, and transwell assays. A combination of MKRN1 quantitative proteomics, ubiquitination modification omics analysis, and a string of in vitro and in vivo experiments revealed the potential mechanisms by which MKRN1 regulates CRC metastasis.ResultsMKRN1 expression was significantly elevated in CRC tissues compared to paracancerous tissues and was positively linked with prognosis (P < 0.01). MKRN1 downregulation inhibits CRC cell proliferation, migration, and invasion. Conversely, MKRN1 overexpression promotes the proliferation, migration, and invasion of CRC cells. Mechanistically, MKRN1 induces epithelial-mesenchymal transition (EMT) in CRC cells via ubiquitination and degradation of Smad nuclear-interacting protein 1 (SNIP1). Furthermore, SNIP1 inhibits transforming growth factor-β (TGF-β) signalling, and MKRN1 promotes TGF-β signalling by degrading SNIP1 to induce EMT in CRC cells. Finally, using conditional knockout mice, intestinal lesions and metastatic liver microlesions were greatly reduced in the intestinal knockout MKRN1 group compared to that in the control group.ConclusionsHigh MKRN1 levels promote TGF-β signalling through ubiquitination and degradation of SNIP1, thereby facilitating CRC metastasis, and supporting MKRN1 as a CRC pro-cancer factor. The MKRN1/SNIP1/TGF-β axis may be a potential therapeutic target in CRC.

Circ-MALAT1 accelerates cell proliferation and epithelial mesenchymal transformation of colorectal cancer through regulating miR-506-3p/KAT6B axis.

Colorectal cancer (CRC) is a prevalent malignant tumor of the digestive tract. Circular RNAs may play important roles in the progression of CRC. In this study, we investigated the roles and mechanisms of action of circ-MALAT1 in CRC. Gene expression and protein abundance were determined using qRT-PCR and western blot, respectively. Cell proliferation and migration were assessed by MTT, clone formation, and wound-healing assays. The interactions among the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (circ-MALAT1), miR-506-3p, and lysine acetyltransferase 6B (KAT6B) were predicted using the StarBase software and confirmed by the luciferase activity assay. Circ-MALAT1 and KAT6B were upregulated, while miR-506-3p was downregulated in CRC cells. We validated that knocking down of circ-MALAT1 suppressed proliferation, migration, and epithelial-mesenchymal transition (EMT) of CRC cells, and these effects were abolished by miR-506-3p downregulation or KAT6B sufficiency. Our study suggests that circ-MALAT1 could sponge miR-506-3p to regulate the expression of KAT6B. Moreover, KAT6B sufficiency could neutralize miR-506-3p-dependent growth arrest, migration, and EMT. Circ-MALAT1 promotes cell proliferation, migration, and EMT of CRC cells via the miR-506-3p/KAT6B axis, thereby acting as a novel potential therapeutic target for the treatment of colorectal cancer.

ADAMDEC1 induces EMT and promotes colorectal cancer cells metastasis by enhancing Wnt/β-catenin signaling via negative modulation of GSK-3β

Colorectal cancer (CRC) is a highly invasive malignant tumor with pronounced proliferation capacity and is prone to epithelial-mesenchymal transition (EMT) and subsequent metastasis. A disintegrin and metalloproteinase domain-like decysin 1 (ADAMDEC1) is a proteolytically active metzincin metalloprotease that is involved in extracellular matrix remodeling, cell adhesion, invasion, and migration. However, the effects of ADAMDEC1 on CRC are unclear. This study was conducted to investigate the expression and biological role of ADAMDEC1 in CRC. We found that ADAMDEC1 was differentially expressed in CRC. Further, ADAMDEC1 was found to enhance CRC proliferation, migration, and invasion while inhibiting apoptosis. Exogenous ADAMDEC1 overexpression elicited EMT in CRC cells, as evidenced by alterations in E-cadherin, N-cadherin, and vimentin expression. In ADAMDEC1 knockdown or ADAMDEC1 overexpressed CRC cells, the western blotting analysis revealed that Wnt/β-catenin signaling pathway-related proteins were down-regulated or up-regulated. Furthermore, an inhibitor of the Wnt/β-catenin pathway (FH535) partially negated the effect of ADAMDEC1 overexpression on EMT and CRC cell proliferation. Further mechanistic research suggested that ADAMDEC1 knockdown may upregulate GSK-3β and inactivate the Wnt/β-catenin pathway, accompanied by suppressing the expression of β-catenin. Additionally, the blocker of GSK-3β (CHIR-99021) markedly abolished the inhibitory effect of ADAMDEC1 knockdown on Wnt/β-catenin signaling. Our results indicate that ADAMDEC1 promotes CRC metastasis by negatively regulating GSK-3β, activating the Wnt/β-catenin signaling pathway, and inducing EMT, presenting its potential as a therapeutic target for the treatment of metastatic CRC.

KLF2 inhibits colorectal cancer progression and metastasis by inducing ferroptosis via the PI3K/AKT signaling pathway.

Krüppel-like factor 2 (KLF2) belongs to the zinc finger family and is thought to be a tumor suppressor gene due to its low expression in various cancer types. However, its functional role and molecular pathway involvement in colorectal cancer (CRC) are not well defined. Herein, we investigated the potential mechanism of KLF2 in CRC cell invasion, migration, and epithelial-mesenchymal transition (EMT). We utilized the TCGA and GEPIA databases to analyze the expression of KLF2 in CRC patients and its correlation with different CRC stages and CRC prognosis. RT-PCR, western blot, and immunohistochemistry assays were used to measure KLF2 expression. Gain-of-function assays were performed to evaluate the role of KLF2 in CRC progression. Moreover, mechanistic experiments were conducted to investigate the molecular mechanism and involved signaling pathways regulated by KLF2. Additionally, we also conducted a xenograft tumor assay to evaluate the role of KLF2 in tumorigenesis. KLF2 expression was low in CRC patient tissues and cell lines, and low expression of KLF2 was associated with poor CRC prognosis. Remarkably, overexpressing KLF2 significantly inhibited the invasion, migration, and EMT capabilities of CRC cells, and tumor growth in xenografts. Mechanistically, KLF2 overexpression induced ferroptosis in CRC cells by regulating glutathione peroxidase 4 expression. Moreover, this KLF2-dependent ferroptosis in CRC cells was mediated by inhibiting the PI3K/AKT signaling pathway that resulted in the suppression of invasion, migration, and EMT of CRC cells. We report for the first time that KLF2 acts as a tumor suppressor in CRC by inducing ferroptosis via inhibiting the PI3K/AKT signaling pathway, thus providing a new direction for CRC prognosis assessment and targeted therapy.

Src-FAK Signaling Mediates Interleukin 6-Induced HCT116 Colorectal Cancer Epithelial–Mesenchymal Transition

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.

A disintegrin and metalloprotease 12 contributes to colorectal cancer metastasis by regulating epithelial‑mesenchymal transition.

A disintegrin and metalloprotease 12 (ADAM12) and epithelial‑mesenchymal transition (EMT) are linked in the metastasis of various types of cancer. The present study aimed to assess the ability of ADAM12 to induce EMT and its potential as a therapeutic target for colorectal cancer (CRC). ADAM12 expression in CRC cell lines, CRC tissues and a mouse model of peritoneal metastasis was assessed. The effect of ADAM12 on CRC EMT and metastasis was investigated using ADAM12‑pcDNA6‑myc and ADAM12‑pGFP‑C‑shLenti constructs. ADAM12 overexpression enhanced the proliferation, migration, invasion and EMT of CRC cells. The phosphorylation levels of factors associated with the PI3K/Akt pathway were also increased by ADAM12 overexpression. The knockdown of ADAM12 reversed these effects. ADAM12 expression and the loss of E‑cadherin expression were significantly associated with poorer survival compared with other expression statuses of both proteins. In a mouse model of peritoneal metastasis, overexpression of ADAM12 induced increased tumor weight and peritoneal carcinomatosis index compared with that in the negative control group. Conversely, knockdown of ADAM12 reversed these effects. Furthermore, E‑cadherin expression was significantly decreased by overexpression of ADAM12 compared with in the negative control group. By contrast, E‑cadherin expression was increased by knockdown of ADAM12 compared with in the negative control group. ADAM12 overexpression contributed to CRC metastasis by regulating EMT. In addition, in the mouse model of peritoneal metastasis, ADAM12 knockdown exhibited strong anti‑metastatic action. Consequently, ADAM12 may be considered a therapeutic target for CRC metastasis.

LINC00543 promotes colorectal cancer metastasis by driving EMT and inducing the M2 polarization of tumor associated macrophages

BackgroundThe interaction between the tumor-microenvironment (TME) and the cancer cells has emerged as a key player in colorectal cancer (CRC) metastasis. A small proportion of CRC cells which undergo epithelial-mesenchymal transition (EMT) facilitate the reshaping of the TME by regulating various cellular ingredients.MethodsImmunohistochemical analysis, RNA immunoprecipitation (RIP), RNA Antisense Purification (RAP), dual luciferase assays were conducted to investigate the biological function and regulation of LINC00543 in CRC. A series in vitro and in vivo experiments were used to clarify the role of LINC00543 in CRC metastasis.ResultsHere we found that the long non-coding RNA LINC00543, was overexpressed in colorectal cancer tissues, which correlated with advanced TNM stage and poorer prognosis of CRC patients. The overexpression of LINC00543 promoted tumorigenesis and metastasis of CRC cells by enhancing EMT and remodeling the TME. Mechanistically, LINC00543 blocked the transport of pre-miR-506-3p across the nuclear-cytoplasmic transporter XPO5, thereby reducing the production of mature miR-506-3p, resulting in the increase in the expression of FOXQ1 and induction of EMT. In addition, upregulation of FOXQ1 induced the expression of CCL2 that accelerated the recruitment of macrophages and their M2 polarization.ConclusionsOur study showed that LINC00543 enhanced EMT of CRC cells through the pre-miR-506-3p/FOXQ1 axis. This resulted in the upregulation of CCL2, leading to macrophages recruitment and M2 polarization, and ultimately stimulating the progression of CRC.

CircCAPRIN1 interacts with STAT2 to promote tumor progression and lipid synthesis via upregulating ACC1 expression in colorectal cancer.

Circular RNAs (circRNAs) generated by back-splicing of precursor mRNAs (pre-mRNAs) are often aberrantly expressed in cancer cells. Accumulating evidence has revealed that circRNAs play a critical role in the progression of several cancers, including colorectal cancer (CRC). However, the current understandings of the emerging functions of circRNAs in CRC lipid metabolism and the underlying molecular mechanisms are still limited. Here, we aimed to explore the role of circCAPRIN1 in regulating CRC lipid metabolism and tumorigenesis. circRNA microarray was performed with three pairs of tumor and non-tumor tissues from CRC patients. The expression of circRNAs were determined by quantitative PCR (qPCR) and in situ hybridization (ISH). The endogenous levels of circRNAs in CRC cells were manipulated by transfection with lentiviruses overexpressing or silencing circRNAs. The regulatory roles of circRNAs in the occurrence of CRC were investigated both in vitro and in vivo using gene expression array, RNA pull-down/mass spectrometry, RNA immunoprecipitation assay, luciferase reporter assay, chromatin immunoprecipitation analysis, and fluorescence in situ hybridization (FISH). Among circRNAs, circCAPRIN1 was most significantly upregulated in CRC tissue specimens. circCAPRIN1 expression was positively correlated with the clinical stage and unfavorable prognosis of CRC patients. Downregulation of circCAPRIN1 suppressed proliferation, migration, and epithelial-mesenchymal transition of CRC cells, while circCAPRIN1 overexpression had opposite effects. RNA sequencing and gene ontology analysis indicated that circCAPRIN1 upregulated the expressions of genes involved in CRC lipid metabolism. Moreover, circCAPRIN1 promoted lipid synthesis by enhancing Acetyl-CoA carboxylase 1 (ACC1) expression. Further mechanistic assays demonstrated that circCAPRIN1 directly bound signal transducer and activator of transcription 2 (STAT2) to activate ACC1 transcription, thus regulating lipid metabolism and facilitating CRC tumorigenesis. These findings revealed the oncogenic role and mechanism of circCAPRIN1 in CRC. circCAPRIN1 interacted with STAT2 to promote CRC tumor progression and lipid synthesis by enhancing the expression of ACC1. circCAPRIN1 may be considered as a novel potential diagnostic and therapeutic target for CRC patients.

Circ_0056618 enhances PRRG4 expression by competitively binding to miR-411-5p to promote the malignant progression of colorectal cancer.

The purpose of this paper was to explore the role of circ_0056618 and associated mechanisms in colorectal cancer (CRC). The expression of circ_0056618, proline rich and Gla domain 4 (PRRG4) mRNA and miR-411-5p was measured by quantitative real-time PCR (qPCR).The protein levels of PRRG4 and epithelial-mesenchymal transition (EMT)-related markers were detected by western blot. Cell proliferation was assessed by cell counting kit-8, EdU, and colony formation assays. Cell migration and invasion were assessed by transwell assay. Cell apoptosis was detected by flow cytometry assay. The putative relationship between miR-411-5p and circ_0056618 or PRRG4 was verified by dual-luciferase reporter assay. The effects of circ_0056618 on tumor growth in vivo were determined by animal study. Circ_0056618 and PRRG4 was upregulated, while miR-411-5p was downregulated in CRC tumor tissues and cells. Circ_0056618 knockdown or PRRG4 knockdown inhibited CRC cell proliferation, migration/invasion, EMT, and survival. Circ_0056618 positively modulated PRRG4 expression by targeting miR-411-5p. MiR-411-5p absence or PRRG4 overexpression could rescue circ_0056618 knockdown-induced inhibition on proliferation, migration/invasion, and EMT in CRC cells. Animal assay showed circ_0056618 knockdown impeded tumor growth in vivo. Circ_0056618 promoted CRC growth and development by upregulating PRRG4 expression via competitively targeting miR-411-5p.

GLUT3 Promotes Epithelial-Mesenchymal Transition via TGF-β/JNK/ATF2 Signaling Pathway in Colorectal Cancer Cells.

Glucose transporter (GLUT) 3, a member of the GLUTs family, is involved in cellular glucose utilization and the first step in glycolysis. GLUT3 is highly expressed in colorectal cancer (CRC) and it leads to poor prognosis to CRC patient outcome. However, the molecular mechanisms of GLUT3 on the epithelial–mesenchymal transition (EMT) process in metastatic CRC is not yet clear. Here, we identified that activation of the c-Jun N-terminal kinase (JNK)/activating transcription factor-2 (ATF2) signaling pathway by transforming growth factor-β (TGF-β) promotes GLUT3-induced EMT in CRC cells. The regulation of GLUT3 expression was significantly associated with EMT-related markers such as E-cadherin, α- smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), vimentin and zinc finger E-box binding homeobox 1 (ZEB1). We also found that GLUT3 accelerated the invasive ability of CRC cells. Mechanistically, TGF-β induced the expression of GLUT3 through the phosphorylation of JNK/ATF2, one of the SMAD-independent pathways. TGF-β induced the expression of GLUT3 by increasing the phosphorylation of JNK, the nuclear translocation of the ATF2 transcription factor, and the binding of ATF2 to the promoter region of GLUT3, which increased EMT in CRC cells. Collectively, our results provide a new comprehensive mechanism that GLUT3 promotes EMT process through the TGF-β/JNK/ATF2 signaling pathway, which could be a potential target for the treatment of metastatic CRC.

Hsa_circ_0006732 regulates colorectal cancer cell proliferation, invasion and EMT by miR-127-5p/RAB3D axis.

Colorectal cancer (CRC) remains a malignancy tumor with high metastasis and poor prognosis. We aimed to explore the effect of circular RNA (circRNA) hsa_circ_0006732 in the progression of CRC. Hsa_circ_0006732 expression in CRC tissues and cell lines were detected using qRT-PCR. The relationship between hsa_circ_0006732 expression and clinicopathologic characteristics of patients with CRC was analyzed. Loss-of-function assay was conducted to determine the regulatory effect of hsa_circ_0006732 on CRC cell proliferation, migration and invasion by using the CCK-8, wound-healing assay and transwell assays. Protein expression changes on epithelial mesenchymal transition (EMT)-related factors were detected by western blotting. The downstream signaling pathway was investigated by bioinformatics, dual-luciferase reporter assay. Rescue assay was further examined for prediction validation. It was found that hsa_circ_0006732 was highly expressed in CRC tissues and cell lines. Downregulation of hsa_circ_0006732 suppressed the proliferation, migration, invasion and EMT of CRC cells. Further mechanistic investigations proved that hsa_circ_0006732 functioned as a competitive endogenous RNA (ceRNA) by directly sponging of miR-127-3p, which further affected the expression of Ras-related protein Rab-3D (Rab3D). Taken together, these findings indicated that hsa_circ_0006732 might be an oncogene in CRC through the regulation of the miR-127-5p/RAB3D axis. Thus, hsa_circ_0006732 might serve as a potential therapeutic target for the treatment of CRC.

Forkhead box A2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced epithelial-mesenchymal transition by targeting c14orf28 in colorectal cancer

Background and Study AimsThis study aimed to investigate the effect of Forkhead Box A2 (FOXA2) on migration, invasion, and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells in hypoxia and explore its related molecular mechanisms. Patients and MethodsA cellular hypoxia model was established, and the FOXA2 overexpression vector was transfected into SW480 and HCT116 cells. Cell apoptosis, migration, and invasion were examined by flow cytometry, scratch test, and transwell-invasion assay. Next, the hsa-let-7 g gene expression was detected by quantitative reverse transcription-polymerase chain reaction. Relative protein levels of HIF-1, FOXA2, c14orf28, E-cadherin, N-cadherin, and Vimentin were detected by western blot. ResultsHypoxia-exposed CRC cells showed a significantly increased cell apoptosis rate, as well as enhanced cell invasion and migration abilities compared with the cells in normoxia. FOXA2 overexpression induced apoptosis and inhibited hypoxia-exposed CRC cell migration and invasion. Additionally, FOXA2 overexpression led to the significantly increased hsa-let-7 g and E-cadherin expression, as well as the decreased c14orf28, N-cadherin, and Vimentin expression in hypoxic CRC cells. ConclusionsThis study demonstrated that FOXA2 could affect the apoptosis, migration, invasion, and EMT of CRC cells under hypoxia conditions. FOXA2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced EMT by targeting c14orf28.

Degradation of HIF-1α induced by curcumol blocks glutaminolysis and inhibits epithelial-mesenchymal transition and invasion in colorectal cancer cells.

Colorectal cancer (CRC) has high morbidity and mortality. Epithelial-mesenchymal transition (EMT) is associated with CRC progression and metastasis. Glutaminolysis is essential for malignancy of cancer cells. Here, we examined the effects of curcumol on CRC EMT. We observed that curcumol suppressed invasion and migration in human CRC cells associated with upregulation of epithelial markers E-cadherin and Zonula occludens 1 and downregulation of mesenchymal markers N-cadherin and Vimentin as well as EMT-related transcription factors Snail and Twist. Curcumol increased intracellular levels of glutamine but decreased intracellular levels of glutamate, α-ketoglutarate, ATP, glutathione, and tricarboxylic acid cycle metabolites, suggesting interruption of glutaminolysis. Next, curcumol repressed glutaminase 1 (Gls1) mRNA and protein expression, and overexpression of Gls1 promoted EMT and abolished curcumol effects on CRC cell EMT. Molecular examinations showed that curcumol stimulated protein degradation of hypoxia-inducible factor-1α (HIF-1α) and prevented its nuclear accumulation in CRC cells. HIF-1α agonist deferoxamine (DFO) promoted HIF-1α binding to Gls1 promoter and increased Gls1 expression but abolished curcumol's inhibitory effects on Gls1 expression. DFO also enhanced EMT and invasion and migration in CRC cells and eliminated curcumol effects. Furthermore, mouse CRC models were established with in vivo overexpression of HIF-1α and Gls1. Curcumol effectively inhibited CRC growth, metastasis, and EMT in mice, which was abrogated by overexpression of HIF-1α or Gls1. Altogether, stimulation of HIF-1α degradation was required for curcumol to disrupt EMT and repress invasion and migration in CRC cells through inhibiting Gls1-mediated glutaminolysis. Curcumol could be a promising candidate for intervention of CRC metastasis. • Curcumol inhibits EMT and blocks glutaminolysis in CRC cells. • Inhibition of Gls1 is required for curcumol blockade of glutaminolysis and EMT. • Curcumol induces HIF-1α degradation leading to inhibition of Gls1 and blockade of glutaminolysis and EMT. • Curcumol suppresses CRC growth and metastasis via inhibiting HIF-1α, glutaminolysis and EMT in mice.

MYSM1 inhibits human colorectal cancer tumorigenesis by activating miR-200 family members/CDH1 and blocking PI3K/AKT signaling

BackgroundHistone epigenetic modification disorder is an important predisposing factor for the occurrence and development of many cancers, including colorectal cancer (CRC). The role of MYSM1, a metalloprotease that deubiquitinates monoubiquitinated histone H2A, in colorectal cancer was identified to evaluate its potential clinical application value.MethodsMYSM1 expression levels in CRC cell lines and tumor tissues were detected, and their associations with patient survival rate and clinical stage were analyzed using databases and tissue microarrays. Gain- and loss-of-function studies were performed to identify the roles of MYSM1 in CRC cell proliferation, apoptosis, cell cycle progression, epithelial-mesenchymal transition (EMT) and metastasis in vitro and in vivo. ChIP, rescue assays and signal pathway verification were conducted for mechanistic study. Immunohistochemistry (IHC) was used to further assess the relationship of MYSM1 with CRC diagnosis and prognosis.ResultsMYSM1 was significantly downregulated and was related to the overall survival (OS) of CRC patients. MYSM1 served as a CRC suppressor by inducing apoptosis and inhibiting cell proliferation, EMT, tumorigenic potential and metastasis. Mechanistically, MYSM1 directly bound to the promoter region of miR-200/CDH1, impaired the enrichment of repressive H2AK119ub1 modification and epigenetically enhanced miR-200/CDH1 expression. Testing of paired CRC patient samples confirmed the positive regulatory relationship between MYSM1 and miR-200/CDH1. Furthermore, silencing MYSM1 stimulated PI3K/AKT signaling and promoted EMT in CRC cells. More importantly, a positive association existed between MYSM1 expression and a favorable CRC prognosis.ConclusionsMYSM1 plays essential suppressive roles in CRC tumorigenesis and is a potential target for reducing CRC progression and distant metastasis.

TAGLN2 promotes the proliferation, invasion, migration and epithelial-mesenchymal transition of colorectal cancer cells by activating STAT3 signaling through ANXA2.

Colorectal cancer (CRC) is one of the leading causes of cancer-associated mortality worldwide and currently ranks third in the USA in terms of prevalence. Transgelin-2 (TAGLN2) was previously reported to serve as a tumor promoter in various types of cancer. The present study aimed to investigate the role of TAGLN2 in the progression of CRC and to determine the potential underlying mechanism. The expression level of TAGLN2 in CRC cells (HCT116, SNU-C1, LoVo and SW480) were first detected by reverse transcription quantitative PCR and western blotting. Following TAGLN2 knockdown through transfection with short hairpin (sh)RNAs against TAGLN2, CRC cell proliferation was determined using Cell Counting Kit-8 and 5′-ethynyl-2′-deoxyuridine assays. Cell migration and invasion were evaluated using wound healing and Transwell assays, respectively. The expression levels of matrix metalloproteinase (MMP)2, MMP9 and proteins associated with epithelial-mesenchymal transition (EMT), including N-cadherin (N-cad), vimentin, zinc finger E-box binding homeobox 2 (ZEB2) and E-cadherin (E-cad), were also evaluated by western blotting. Furthermore, following TAGLN2 overexpression and the use of signal transducer and activator of transcription 3 (STAT3) inhibitors to treat CRC cells, all the aforementioned biological parameters were evaluated. The potential relationship between annexin 2 (ANXA2) and STAT3 was confirmed by western blotting analysis. The expression level of TAGLN2 was found to be particularly high in CRC cells. Following TAGLN2 knockdown, CRC cell proliferation, migration, invasion and EMT were significantly inhibited. TAGLN2 knockdown also suppressed STAT3 phosphorylation in CRC cells. In addition, the promoting effects of TAGLN2 overexpression on the progression of CRC were reversed by STAT3 inhibitor. Furthermore, ANXA2 was positively associated with STAT3. Taken together, these findings demonstrated that TAGLN2 could promote the proliferation, invasion, migration and EMT of CRC cells by activating STAT3 and regulating ANXA2 expression. This may reveal the underlying mechanism by which TAGLN2 might regulate the progression of CRC and provide potential therapeutic targets for the treatment of CRC.

Tetrandrine Inhibits Epithelial-Mesenchymal Transition in IL-6-Induced HCT116 Human Colorectal Cancer Cells.

IntroductionPatients with colorectal cancer (CRC) often develop distant metastases, which significantly reduces the 5-year survival rate. Epithelial-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of cancer cells. Tetrandrine has been reported to inhibit the viability and EMT of CRC cells; however, to the best of our knowledge, the molecular mechanism remains undetermined.MethodsThe MTT assay was used to determine HCT116 cell viability. Wound healing and Transwell assays were used to determine that cell migration and invasion, respectively. Western blotting analysis was performed to detect the expression of migration-related genes. Four different lengths of the E-cadherin gene promoter were constructed and cloned into pGL3 reporter plasmids to evaluate E-cadherin gene promoter activity.ResultsThe results of the MTT assay revealed that tetrandrine inhibited HCT116 cell viability, with an IC50 value of 7.2 μM following 24 h of treatment. Tetrandrine inhibited IL-6-induced cell migration and invasion, respectively. Tetrandrine regulates the expression of migration-related genes in IL-6-stimulated HCT116 cells. Tetrandrine significantly downregulated the expression and enzyme activity of MMP-2 in IL-6-stimulated HCT116 cells. In addition, tetrandrine restored E-cadherin gene promoter activity.ConclusionThe findings of the present study suggested that tetrandrine may inhibit EMT in IL-6-stimulated HCT116 cells; therefore, it may represent a potential drug for CRC.

Evidence That Tumor Microenvironment Initiates Epithelial-To-Mesenchymal Transition and Calebin A can Suppress it in Colorectal Cancer Cells.

Background: Tumor microenvironment (TME) has a pivotal impact on tumor progression, and epithelial-mesenchymal transition (EMT) is an extremely crucial initial event in the metastatic process in colorectal cancer (CRC) that is not yet fully understood. Calebin A (an ingredient in Curcuma longa) has been shown to repress CRC tumor growth. However, whether Calebin A is able to abrogate TME-induced EMT in CRC was investigated based on the underlying pathways. Methods: CRC cell lines (HCT116, RKO) were exposed with Calebin A and/or a FAK inhibitor, cytochalasin D (CD) to investigate the action of Calebin A in TME-induced EMT-related tumor progression. Results: TME induced viability, proliferation, and increased invasiveness in 3D-alginate CRC cultures. In addition, TME stimulated stabilization of the master EMT-related transcription factor (Slug), which was accompanied by changes in the expression patterns of EMT-associated biomarkers. Moreover, TME resulted in stimulation of NF-κB, TGF-β1, and FAK signaling pathways. However, these effects were dramatically reduced by Calebin A, comparable to FAK inhibitor or CD. Finally, TME induced a functional association between NF-κB and Slug, suggesting that a synergistic interaction between the two transcription factors is required for initiation of EMT and tumor cell invasion, whereas Calebin A strongly inhibited this binding and subsequent CRC cell migration. Conclusion: We propose for the first time that Calebin A modulates TME-induced EMT in CRC cells, at least partially through the NF-κB/Slug axis, TGF-β1, and FAK signaling. Thus, Calebin A appears to be a potential agent for the prevention and management of CRC.

Curcumin inhibits epithelial-mesenchymal transition in colorectal cancer cells by regulating miR-206/SNAI2 pathway

Purpose: To examine the effects of curcumin on epithelial-mesenchymal transition (EMT) via regulation of miR-206 and SNAI2 in colorectal cancer (CRC) cells. Relationship between SNAI2 and miR-206 and the effects of curcumin on related mechanisms were also identified.&#x0D; Methods: Transwell assays were used to analyze cellular migration and invasion. Genes associated with changes in protein and mRNA expression were evaluated by western blotting and quantitative reverse transcription PCR analyses, respectively. The relationship between SNAI2 and miR-206 was determined using a dual luciferase assay.&#x0D; Results: Curcumin inhibited cell metastasis, upregulated miR-206 expression, and decreased SNAI2 levels. Furthermore, miR-206 directly targeted SNAI2 and inhibited EMT via downregulation of SNAI2 expression. Curcumin inhibited EMT in CRC cells by upregulating miR-206.&#x0D; Conclusion: This study, for the first time, discovered the role of curcumin on epithelial-mesenchymal transition process in colorectal cancer cells by modulating miR-206/SNAI2 axis. These findings suggest that curcumin may be useful as a novel therapeutic agent to inhibit the metastasis of CRC.